This proposal is the continuation of our studies on second messenger systems derived from membrane lipids involved in retinal pigment epithelium - visual cell interactions during photoreceptor renewal. We have developed new approaches and a hypothesis that entails a chain of events triggered at the initiation of shedding and phagocytosis. The focus is on the testing of the hypothesis that an initial step in shedding and phagocytosis is the activation of a phospholipase C that generates inositol trisphosphate and diacylglycerol in the retinal pigment epithelial cells. These second messengers then promote intracellular Ca2+ mobilization and protein kinase C activation, respectively, which in turn modulate several cell functions. The ionized Ca2+ then activates both a phospholipase A2, leading to arachidonic acid release, and lipoxygenase enzymes, resulting in formation of leukotrienes (e.g. leukotriene C4) and other metabolites (e.g. 12-hydroxyeicosatetraenoic acid, lipoxins). These messengers diffuse to the interphotoreceptor matrix and are prime candidates as intercellular signals possibly acting on photoreceptor cells. Phospholipase A2 also gives rise to another lipid mediator, platelet-activating factor, which may contribute to the regulation of long-term events in the retinal pigment epithelium. The proposed experimental design will use powerful now analytical procedures, such as high-performance liquid chromatography-particle beam-mass spectrometry and high-performance liquid chromatography-capillary gas-liquid chromatography. These methods will also allow the isolation and identification of storage sites at membranes of lipid mediators, such as 1 -alkyl-2-- arachidonoyl-glycerophosphorylcholine (the precursor of platelet-activating factor) and of the second messengers of the enzymatic oxygenation of arachidonic acid. Frogs will be used because RPE - visual cell interaction can be experimentally manipulated by altering the light-dark cycle, and shedding can be triggered at the onset of light. A rapid procedure to isolate intact retinal pigment epithelial cells will also allow the direct biochemical study in vivo of each step of the hypothesis. This information will be correlated with the histological assessment of shedding and phagocytosis. Moreover, eyecups will be incubated in vitro with selective antagonists or inhibitors of the second messenger systems involved in these events, as well as receptor antagonists to study the shedding response. In addition, human retinal pigment epithelial cells in culture will be used to explore the various steps of the hypothesis. The results will define the involvement of second messengers in the interactions between visual cells and retinal pigment epithelium during photoreceptor renewal.